p16INK4 is a potential target for inactivation in the development of cancer because it regulates passage through the G1 phase of the cell cycle by inhibiting the cyclin-dependent-kinases CDK4 and CDK6. p16INK4 mutations occur frequently in human tumor cell lines3,4 and in several primary tumors. Previously, we described 8 germline p16INK4 mutations in 18 familial melanoma kindreds. Genetic analyses suggested that five mutations (1nonsense, 1 splice donor site, and 3 missense) predisposed carriers to melanoma, and that two missense mutations had no phenotypic effect. In the past year, we have described biochemical analyses of the missense germline mutations and a single somatic mutation detected in these families. The melanoma-associated mutant proteins showed reduced binding to CDK4, and were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 complex in vitro. These data provide a biochemical rationale for the hypothesis that carriers of certain p16INK4 mutations are at greatly increased risk of developing melanoma.